The term “opioid” generically refers to all drugs, natural and synthetic, that have morphine-like actions. Formerly, the term “opiate” was used to designate drugs derived from opium, e.g., morphine, codeine, and many semi-synthetic congeners of morphine. After the isolation of peptide compounds with morphine-like actions, the term opioid was introduced to refer generically to all drugs with morphine-like actions. Included among opioids are various peptides that exhibit morphine-like activity, such as endorphins, enkephalins and dynorphins. However, some sources have continued to use the term “opiate” in a generic sense, and in such contexts, opiate and opioid are interchangeable. Additionally, the term opioid has been used to refer to antagonists of morphine-like drugs as well as to characterize receptors or binding sites that combine with such agents.
Opioids are generally employed as analgesics, but they may have many other pharmacological effects as well. Morphine and related opioids produce their major effects on the central nervous and digestive systems. The effects are diverse, including analgesia, drowsiness, mood changes, respiratory depression, dizziness, mental clouding, dysphoria, pruritus, increased pressure in the biliary tract, decreased gastrointestinal motility, nausea, vomiting, and alterations of the endocrine and autonomic nervous systems.
A significant feature of the analgesia produced by opioids is that it occurs without loss of consciousness. When therapeutic doses of morphine are given to patients with pain, they report that the pain is less intense, less discomforting, or entirely gone. In addition to experiencing relief of distress, some patients experience euphoria. However, when morphine in a selected pain-relieving dose is given to a pain-free individual, the experience is not always pleasant; nausea is common, and vomiting may also occur. Drowsiness, inability to concentrate, difficulty in mentation, apathy, lessened physical activity, reduced visual acuity, and lethargy may ensue.
The development of tolerance and physical dependence with repeated use is a characteristic feature of all opioid drugs, and the possibility of developing psychological dependence on the effect of these drugs is a major limitation for their clinical use. There is evidence that phosphorylation may be associated with tolerance in selected cell populations (Louie, A. et al. Biochem Biophys Res Comm (1988) 152:1369-75).
Acute opioid poisoning may result from clinical overdosage, accidental overdosage, or attempted suicide. In a clinical setting, the triad of coma, pinpoint pupils, and depressed respiration suggest opioid poisoning. Mixed poisonings including agents such as barbiturates or alcohol may also contribute to the clinical picture of acute opioid poisoning. In any scenario of opioid poisoning, treatment must be administered promptly.
The opioids interact with what appear to be several closely related receptors. Various inferences have been drawn from data that have attempted to correlate pharmacologic effects with the interactions of opioids with a particular constellation of opioid receptors (Goodman and Gilman's, THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th ed, pp. 493-95 (MacMillan 1985)). For example, analgesia has been associated with mu and kappa receptors. Delta receptors are believed to be involved in alterations of affective behavior, based primarily on the localization of these receptors in limbic regions of the brain. Additionally, activation, e.g., ligand binding with stimulation of further receptor-mediated responses, of delta opioid receptors is believed to inhibit the release of other neurotransmitters. The pathways containing relatively high populations of delta opioid receptor are similar to the pathways implicated to be involved in Huntington's disease. Accordingly, it is postulated that Huntington's disease may correlate with some effect on delta opioid receptors.
Two distinct classes of opioid molecules can bind opioid receptors: the opioid peptides (e.g., the enkephalins, dynorphins, and endorphins) and the alkaloid opiates (e.g., morphine, etorphine, diprenorphine and naloxone). Subsequent to the initial demonstration of opiate binding sites (Pert, C. B. and Snyder, S. H., Science (1973) 179:1011-1014), the differential pharmacological and physiological effects of both opioid peptide analogues and alkaloid opiates served to delineate multiple opioid receptors. Accordingly, three anatomically and pharmacologically distinct opioid receptor types have been described: delta, kappa and mu. Furthermore, each type is believed to have sub-types (Wollemann, M., J Neurochem (1990) 54:1095-1101; Lord, J. A., et al., Nature (1977) 267:495-499).
All three of these opioid receptor types appear share the same functional mechanisms at a cellular level. For example, the opioid receptors cause inhibition of adenylate cyclase, and inhibition of neurotransmitter release via both potassium channel activation and inhibition of Ca2+ channels (Evans, C. J., In: Biological Basis of Substance Abuse, S. G. Korenman & J. D. Barchas, eds., Oxford University Press (in press); North, A. R., et al., Proc Natl Acad Sci USA (1990) 87:7025-29; Gross, R. A., et al., Proc Natl Acad Sci USA (1990) 87:7025-29; Sharma, S. K., et al., Proc Natl Acad Sci USA (1975) 72:3092-96). Although the functional mechanisms are the same, the behavioral manifestations of receptor-selective drugs differ greatly (Gilbert, P. E. & Martin, W. R., J Pharmacol Exp Ther (1976) 198:66-82). Such differences may be attributable in part to the anatomical location of the different receptors.
Delta receptors have a more discrete distribution within the mammalian CNS than either mu or kappa receptors, with high concentrations in the amygdaloid complex, striatum, substantia nigra, olfactory bulb, olfactory tubercles, hippocampal formation, and the cerebral cortex (Mansour, A., et al., Trends in Neurosci (1988) 11:308-14). The rat cerebellum is remarkably devoid of opioid receptors including delta opioid receptors.
Several opioid molecules are known to selectively or preferentially bind delta receptors. Of the vertebrate endogenous opioids, the enkephalins, particularly met-enkephalin (SEQ ID NO: 1) and leu-enkephalin (SEQ ID NO: 2), appear to possess the highest affinity for delta receptors, although the enkephalins also have high affinity for mu receptors. Additionally, the deltorphans, peptides isolated from frog skin, comprise a family of opioid peptides that have high affinity and selectivity for delta receptors (Erspamer, V., et al., Proc Natl Acad Sci USA (1989) 86:5188-92).
A number of synthetic enkephalin analogues are also delta receptor-selective including (D-Ser2) leucine enkephalin Thr (DSLET) (SEQ ID NO: 3) (Garcel, G. et al. FEBS Lett (1980) 118:245-247) and (D-Pen2, D-Pen5) enkephalin (DPDPE) (SEQ ID NO: 4) (Akiyama, K. et al., Proc Natl Acad Sci USA (1985) 82:2543-2547).
Recently a number of other selective delta receptor ligands have been synthesized, and their bioactivities and binding characteristics suggest the existence of more than one delta receptor subtype (Takemori, A. E., et al., Ann Rev Pharm Toxicol, (1992) 32:239-69; Negri, L., et al., Eur J Pharmacol (1991) 196:355-335; Sofuoglu, M., et al., Pharmacologist (1990) 32:151).
Although the synthetic pentapeptide 2dAla, 5dLeu enkephalin (DADLE) was considered to be delta-selective, it also binds equally well to mu receptors. The synthetic peptide D-Ala2-N-Me-Phe4-Gly-ol5-enkephalin (DAGO) (SEQ ID NO: 6) has been found to be a selective ligand for mu-receptors.
The existence of multiple delta opioid receptors has been implied not only from the pharmacological studies addressed above, but also from molecular weight estimates obtained by use of irreversible affinity ligands. Molecular weights for the delta opioid receptor that range from 30 kDa to 60 kda (Evans, C. J., supra; Evans, C. J. et al., Science (1992) 258:1952-1955, which document corresponds to the disclosure of the priority document of the present application; Bochet, P. et al., Mol Pharmacol (1988) 34:436-43). The various receptor sizes may represent alternative splice products, although this has not been established.
Many studies of the delta opioid receptor have been performed with the neuroblastoma/glioma cell line NG108-15, which was generated by fusion of the rat glial cell line (C6BU-1) and the mouse neuroblastoma cell line (N18-TG2) (Klee, W. A. and Nirenberg, M. A., Proc Natl Acad Sci USA (1974) 71:3474-3477). The rat glial cell line expresses essentially no delta opioid receptors, whereas the mouse neuroblastoma cell line expresses low amounts of the receptor. Thus, it has been suggested that the delta receptor in the NG108-15 cells is of mouse chromosomal origin (Law, Mol Pharm (1982) 21:438-91). Each NG108-15 cell is estimated to express approximately 300,000 delta-receptors. Only delta-type opioid receptors are expressed, although it is not known whether these represent more than a single subtype. Thus, the NG108-15 cell line has served to provide considerable insight into the binding characterization of opioid receptors, particularly delta opioid receptors. However, the NG108-15 cell line is a cancer-hybrid and may not be completely representative of the delta receptor in endogenous neurons due to the unique cellular environment in the hybrid cells.
An extensive literature has argued that the opioid receptors are coupled to G-proteins (see, e.g., Schofield, P. R., et al., EMBO J (1989) 8:489-95), and are thus members of the family of G-protein coupled receptors. G-proteins are guanine nucleotide binding proteins that couple the extracellular signals received by cell surface receptors to various intracellular second messenger systems. Identified members of the G-protein-coupled family share a number of structural features, the most highly conserved being seven apparent membrane-spanning regions, which are highly homologous among the members of this family (Strosberg, A. D., Eur J Biochem (1991) 196:1-10). Evidence that the opioid receptors are members of this family includes the stimulation of GTPase activity by opioids, the observation that GTP analogues dramatically effect opioid and opiate agonist binding, and the observation that pertussis toxin (which by ADP ribosylation selectively inactivates both the Gi and Go subfamilies of G-proteins) blocks opioid receptor coupling to adenylate cyclase and to K+ and Ca2+ channels (Evans, C. J., supra).
The members of the G-protein-coupled receptor family exhibit a range of characteristics. Many of the G-protein-coupled receptors, e.g., the somatostatin receptor and the angiotensin receptor, have a single exon that encodes the entire protein coding region (Strosberg supra; Langord, K., et al., Biochem Biophys Res Comm (1992) 138:1025-1032). However, other receptors, such as substance P receptor and the dopamine D-2 receptor, contain the protein coding region. The D-2 receptor is particularly interesting in that alternate splicing of the gene gives rise to different transcribed products (i.e., receptors) (Evans, C. J., supra; Strosberg, supra). Interestingly, somatostatin ligands are reported to bind to opioid receptors (Terenius, L., Eur J Pharmacol (1976) 38:211; Mulder, A. H., et al., Eur J Pharmacol (1991) 205:1-6) and, furthermore, to have similar molecular mechanisms (Tsunoo, A., et al., Proc Natl Acad Sci USA (1986) 83:9832-9836).
In previous efforts to describe and purify opioid receptors, two clones have been described that were hypothesized either to encode a portion of or entire opioid receptors. The first clone, which encodes the opiate binding protein OBCAM (Schofield et al., supra), was obtained by utilizing a probe designed from an amino acid sequence of a protein purified on a morphine affinity column. OBCAM lacks any membrane spanning domains but does have a C-terminal domain that is characteristic of attachment of the protein to the membrane by a phosphatidylinositol (PI) linkage. This feature, which is shared by members of the immunoglobulin superfamily, is not common to the family of receptors coupled to G-proteins. Thus, it has been proposed that OBCAM is part of a receptor complex along with other components that are coupled to G-proteins (Schofield et al., supra). At present, however, there is no direct evidence for such a complex.
A second proposed opioid receptor clone was obtained in an effort to clone a receptor that binds kappa opioid receptor ligands (Xie, G. X., Proc Natl Acad Sci USA (1992) 89:4124-4128). A DNA molecule encoding a G-coupled receptor from a placental cDNA library was isolated. This receptor has an extremely high homology with the neurokinin B receptor (84% identical throughout the proposed protein sequence). When this clone was expressed in COS cells, it displayed opioid peptide displaceable binding of 3H-bremazocine (an opiate ligand with high affinity for kappa receptors). However, the low affinity of this receptor for 3H-bremazocine, and the lack of appropriate selectivity since this receptor (binding both mu and delta ligands) made it doubtful that this cloned molecule is actually an opioid receptor.
Furthermore, characterization of opioid receptor proteins has proven difficult because of their instability once solubilized from the membrane; purified delta opioid receptors have not been isolated. The previous estimates of opioid receptor molecular weights ranging from 30 kDa to 60 Kda further reflect the difficulty in isolating and characterizing these proteins.
Recently, DNA encoding the murine kappa and delta opioid receptors from mouse brain was reported by Yasuda, K. et al. Proc Natl Acad Sci USA (1993) 90:6736-6740. The sequence of the clones indicated the presence of the expected seven transmembrane regions. In addition, Chen, Y. et al. in a soon-to-be-published manuscript in Molecular Pharmacology (1993) report the “molecular cloning and functional expression of a mu opioid receptor from rat brain”. In fact, the rat mu receptor was cloned using the present inventors' DOR-1 clone, which lends enabling support to the present invention disclosed below. The mouse delta opioid receptor was disclosed as having been cloned (Kieffer, B. J. et al., Proc Natl Acad Sci USA (1992) 89:12048-12052 (December issue) after the filing date of the priority document of the present application. However, the sequence reported therein differs from the sequence reported by the present inventors for the mouse delta receptor (Evans et al., 1992, supra; this disclosure).
In addition to the opioid receptors which respond to specified agonists, the delta, kappa and mu opioid receptors, additional forms of these proteins, commonly called opioid receptor-like (ORL) proteins have been obtained using the methods described herein. Using these methods, two human ORL protein-encoding cDNAs were obtained from a human brain stem cDNA library. One of these clones is equivalent to that isolated by O'Dowd, B. F. et al. Gene (1993) 136:355-360; the other, ORL-1, is identical to that reported by Mollereau, C. et al. FEBS Lett (1994) 341:33-38. A preliminary report of the present work appeared in Regulatory Peptides (1994) 54:143-144 and is incorporated herein by reference.